HaloLink Protein Array Systems Technical Manual #TM310/media/files/resources/protocols...C....

T e c h n i c a l M a n u a l

HaloLink™ Protein Array SystemsINSTRUCTIONS FOR USE OF PRODUCT G6140, G6180 AND G6190.

PRINTED IN USA. Revised 12/15 Part# TM310

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA. Part# TM310Revised 12/15 Page 1

1. Description ....................................................................................................................................................................1

2. Product Components and Storage Conditions.......................................................................................................4

3. Required Materials and Experimental Considerations ........................................................................................6

4. Protocol for Expression and Capture of HaloTag® Fusion Proteins to Create a Custom Protein Array ...7A. Expressing HaloTag® Fusion Proteins.............................................................................................................9B. Confirming Expression of HaloTag® Fusion Proteins with an SDS-Gel (optional) ...............................11C. Attaching the HaloLink™ Array Gasket to the HaloLink™ Slide ...........................................................12D. Immobilization of HaloTag® Fusion Proteins on HaloLink™ Slides .......................................................15E. Confirming Capture of HaloTag® Fusion Proteins on HaloLink™ Slides (optional)...........................17F. Using HaloTag® Standard Protein for Normalization (optional) .............................................................18

5. Guidelines for Protein:Protein Interaction Analysis .........................................................................................19A. Expression and Cotranslational Labeling of Probe Protein in a Cell-Free System ................................21B. Protein:Protein Interaction Analysis..............................................................................................................23

6. Guidelines for Protein:DNA Interaction Analysis ............................................................................................23

7. Scanning and Data Analysis of HaloLink™ Slides ............................................................................................25A. Scanning .............................................................................................................................................................25B. Data Analysis.....................................................................................................................................................26

8. Troubleshooting .........................................................................................................................................................27

9. References ....................................................................................................................................................................28

10. Appendix......................................................................................................................................................................29A. HaloTag® Vectors..............................................................................................................................................29B. Composition of Buffers and Solutions ..........................................................................................................29C. Related Products ...............................................................................................................................................30D. Summary of Change.........................................................................................................................................30

1. Description

Protein arrays enable proteome-scale detection of protein:protein, protein:drug or protein:nucleic acidinteractions. The HaloLink™ Protein Array Systems(a,b) provide a new way to create custom proteinarrays by combining innovative HaloTag® technology, surface engineering and cell-free proteinexpression systems.

All technical literature is available on the Internet at: www.promega.com/protocols/Please visit the web site to verify that you are using the most current version of this Technical Manual.

Please contact Promega Technical Services if you have questions on the use of this product. Email: [email protected]

HaloLink™ Protein Array Systems

1. Description (continued)

The HaloTag® protein is a mutated hydrolase that forms a covalent bond with HaloTag® ligands. Underphysiological conditions binding is rapid and highly specific, yielding a complex that is stable evenunder stringent conditions (1–3). Using the HaloLink™ Protein Array Systems, HaloTag® fusionproteins are expressed in a cell-free expression system and then captured on hydrogel-coated glassslides. The fusion proteins are captured directly from the expression reaction mixture without priorpurification. Using this approach, multiple fusion proteins can be rapidly synthesized and immobilizedin parallel on the slide surface, and a complete experiment including protein expression, custom arrayformation, and protein interaction analysis can be completed in less than eight hours.

Features of the HaloLink™ Protein Array Systems

• Fast protein expression: Cell-free expression systems allow quick, single-tube, coupledtranscription/translation.

• Irreversible binding of the captured protein: Unlike other affinity tags, which tend to dissociate fromthe surface, HaloTag® fusion proteins are covalently bound to the HaloLink™ Slide.

• No protein purification: The protein of interest is immobilized directly from the cell-free expressionsystem.

• Reduced nonspecific binding: HaloLink™ Slides have a unique hydrogel coating that is designed toprevent nonspecific binding while preserving the functionality of specifically captured proteins.

• Extensive washing: Covalent binding of HaloTag® fusion proteins to the HaloLink™ Slide allowsextensive, stringent washing, that may result in reduced background and a lower incidence of falsepositives.

• No need for a robotic arrayer: The unique 50-well configuration allows multiple interactions to bestudied in parallel without the need for a complex robotic arrayer.

HaloLink™ Protein Array Systems Overview (Figure 1)

1. The desired protein-coding sequence is cloned into an appropriate HaloTag® Flexi® Vector.

2. HaloTag® fusion proteins are expressed in a cell-free expression system.

3. The supplied HaloLink™ Array Gasket is applied to the HaloLink™ Slide, creating 50 leak-free wells.

4. The cell-free expression reaction products are applied and captured on the HaloLink™ Slide, creatinga custom array.

5. The array is designed for downstream applications such as protein:protein interactions and may alsohave potential for protein:DNA interactions, antibody screening, and enzymatic functional analysis.

6. Experimental workflow is provided in Figure 2.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TM310 Printed in USA.Page 2 Revised 12/15


Figure 1. HaloLink™ Protein Array Systems overview.

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Custom array: HaloLink™ Slidewith various HaloTag® fusion proteins

Phase 1 Phase 2Attach gasket toHaloLink™ Slide

HaloLink™ SlideAttach HaloLink™ Array Gasket

HaloLink™ Slidewith 50 wells

Phase 3Immobilizing HaloTag® fusion proteins

onto HaloLink™ Slide

Add cell-free reaction to individual wells

Incubate for 1 hour

Wash and spin dry

Applications

Add probe

Identify interactions

Cell-free synthesisof HaloTag® fusion

proteins

1. Description (continued)

The HaloLink™ Array Two Slide Systems (Cat.# G6140 and G6180) contain HaloLink™ Slides,HaloLink™ Array Gaskets for creating 50-well arrays, a HaloTag® Standard Protein, Anti-HaloTag®

Antibody and a cell-free expression system [either the TNT® T7 Quick Coupled Transcription/Translation System (Cat.# G6140) or the TNT® SP6 High-Yield Wheat Germ Protein Expression System(Cat.# G6180)].

The HaloLink™ Array Six Slide System contains HaloLink™ Slides, HaloLink™ Array Gaskets andAnti-HaloTag® Antibody. Users of the Six Slide System will need either to provide their own proteinexpression system or order the TNT® T7 Quick Coupled Transcription/Translation System (Cat.# L1170and L1171) or TNT® SP6 High Yield Wheat Germ Protein Expression System (Cat.# L3260 and L3261).The HaloTag® Standard Protein (Cat.# G4491) is not included with the Six Slide System but can beordered separately. The HaloLink™ Array Systems were developed using the TNT® T7 Quick and TNT®

SP6 High-Yield Wheat Germ Systems. If you choose to use a different protein expression system, youwill need to optimize the experimental conditions appropriately.

This Technical Manual contains a detailed protocol for use of the HaloLink™ Protein Array Systems tocreate custom arrays and provides guidelines for performing protein:protein (Section 5) andprotein:DNA interaction (Section 6) applications.

2. Product Components and Storage Conditions

Product Cat.#HaloLink™ Array (TNT® T7 Quick) Two Slide System G6140G6140 consists of two parts, which are shipped separately. Includes:

Part 1 of 2 (G6141) HaloLink™ Array (TNT® T7 Quick) Two Slide System

• 3 × 200µl TNT® T7 Quick Master Mix • 1.25ml Nuclease-Free Water• 50µl Methionine• 30µg HaloTag® Standard Protein (3.0mg/ml)• 20µg Anti-HaloTag® pAb (1.0mg/ml)

Part 2 of 2 (G4941) HaloLink™ Slides• 2 HaloLink™ Slides• 2 HaloLink™ Array Gaskets



Product Cat.#HaloLink ™ Array (TNT® SP6 Wheat Germ) Two Slide System G6180G6180 consists of two parts, which are shipped separately. Includes:

Part 1 of 2 (G6181) HaloLink™ Array (TNT® SP6 Wheat Germ) Two Slide System• 2 × 300µl TNT® SP6 High-Yield Wheat Germ Master Mix• 1.25ml Nuclease Free Water• 30µg HaloTag® Standard Protein (3.0mg/ml)• 20µg Anti-HaloTag® pAb (1.0mg/ml)

Part 2 of 2 (G4941) HaloLink™ Slides• 2 HaloLink™ Slides• 2 HaloLink™ Array Gaskets

Product Cat.#HaloLink™ Array Six Slide System G6190Includes:

• 6 HaloLink™ Slides • 6 HaloLink™ Array Gaskets• 20µg Anti HaloTag® pAb (1.0mg/ml)

Storage Conditions: Store the TNT® lysates at –70°C. Store the HaloTag® Standard Protein and theAnti-HaloTag® Antibody at –20°C. The HaloLink™ Protein Array Slides should be stored at –20°C andopened just before use. After opening, unused slides should be stored at –20°C and used within onemonth. Store the HaloLink™ Array Gaskets at room temperature.

• Do not store the TNT® lysate at any temperature other than –70°C. Storage at other temperatures (e.g.,–20°C) for even a short time will dramatically reduce activity.

• Do not freeze-thaw the TNT® lysate more than two times.

• Do not store the TNT® lysate in the presence of dry ice. Prolonged exposure to dry ice can causesignificant loss of activity.

3. Required Materials and Experimental Considerations

HaloTag® Vector (not supplied in the kit)

Before using the HaloLink™ Array System to create custom arrays, the desired protein coding sequencemust be cloned into a HaloTag® Flexi® Vector. See the Appendix (Section 10.A) for specificrecommendations on HaloTag® Vectors available for use with this system.

Important: If your application requires a specificity control (HaloTag® Protein without a fusion partner),please contact Promega Technical Services for information on how to create a construct that expressesHaloTag® Protein alone.Note: Difference between HaloTag® Flexi® Vectors and discontinued pHT2 Vector (Cat.# G8241). TheHaloTag® protien encoded by the HaloTag® Flexi® Vectors is HaloTag® 7, which is the second-generation commerically available HaloTag® protein. It provides increase stability with regard to bothtemperature and denaturants, increased solubility and faster labeling kinetics, resulting in markedlyimproved expression compared to the HaloTag® 2 protein contained in the pHT2 HaloTag® Vector. Thediscontinued pHT2 Vector is not recommended for expression in bacterial cells.

Cell-Free Expression Systems

The TNT® T7 Quick Master Mix (supplied with Cat.# G6140) provides a single-tube, rabbit reticulocytelysate-based eukaryotic coupled transcription/translation system designed to express 1–10µg/ml of protein.To use this system, 0.2–2.0µg of circular plasmid DNA containing a T7 promoter is added to an aliquot of theTNT® T7 Quick Master Mix and incubated in a 50µl reaction volume for 60–90 minutes at 30°C.

The TNT® SP6 High-Yield Wheat Germ Master Mix (supplied with Cat.# G6180) provides a single-tube,coupled transcription/translation reaction designed to express up to 100µg/ml of protein in two hours.To use this system, 2–12µg of circular plasmid DNA containing an SP6 promoter is added to an aliquotof the TNT® SP6 High-Yield Wheat Germ Master Mix and incubated in a 50µl reaction volume for twohours at 25°C.

We strongly recommend that you refer to the TNT® T7 Quick Coupled Transcription/Translation SystemsTechnical Manual #TM045 (www.promega.com/tbs/tm045/tm045.html) or the TNT® SP6 High-YieldWheat Germ Protein Expression System Technical Manual # TM282 (www.promega.com/tbs/) for specificdetails on use of these expression systems.

The simplicity of cell-free reactions performed using the TNT® Systems means that multiple HaloTag®

fusion proteins can be expressed in parallel reactions. Other cell-free protein expression systems, as wellas in vivo protein expression in mammalian cells or E. coli, can be used for expressing HaloTag® fusionproteins for use with HaloLink™ Protein Arrays, but the user will need to optimize any otherexpression system.

HaloLink™ Slides (supplied with all kits)

The HaloLink™ Slides are designed for protein applications. Each slide has an organic hydrogel coatingactivated with HaloTag® Ligand that:• Inhibits nonspecific binding of proteins • Specifically captures HaloTag® fusion proteins• Provides an ideal environment for maintaining the functional integrity of captured proteins



HaloLink™ Array Gaskets (supplied with all kits)

The HaloLink™ Array Gaskets are used to make 50 wells on the HaloLink™ Slides. The gasket is attachedby placing the HaloLink™ Slide onto the gasket and gently pressing to make leak-proof wells that eliminatewell-to-well cross contamination. Each well is 3.0mm in diameter and 1.0mm in height and has a capacity of10µl. The gasket can be removed at the end of the assay for scanning and quantitating the signal.

HaloTag® Standard Protein (supplied with Cat.# G6140 and G6180) and Anti-HaloTag® pAb(supplied with all kits)

The HaloTag® Standard Protein is a 61kDa purified HaloTag®-GST fusion protein supplied at aconcentration of 3.0mg/ml. HaloTag® Standard Protein can be used to estimate the expression level ofyour HaloTag® fusion protein. Typical concentration range of the HaloTag® Standard Protein to use willdepend on the cell-free expression system. With TNT® T7 Quick reactions, the concentration range is1–10µg/ml (0.016–0.16 nmol/ml). With TNT® SP6 High-Yield Wheat Germ reactions, it is 10–100µg/ml(0.16–1.6 nmol/ml).

The Anti-HaloTag® pAb is a purified rabbit polyclonal antibody raised against the HaloTag® protein.The antibody is supplied at a concentration of 1.0mg/ml in PBS.

Anti-HaloTag® pAb and HaloTag® Standard Protein can be used to confirm and estimate expressionlevels of HaloTag® fusion proteins in cell-free systems as well as to confirm and estimate capture of theHaloTag® fusion proteins on the slide surface and to normalize for variations in:• Day-to-day expression levels• Expression levels in different systems• Expression levels of panels of HaloTag® fusion proteins• Capture efficiency of different proteins

4. Protocol for Expression and Capture of HaloTag® Fusion Proteins to Create a Custom Protein Array

This section contains a protocol for expression of HaloTag® fusion proteins in cell-free systems andsubsequent capture of these fusion proteins onto HaloLink™ Slides. A detailed workflow is shown inFigure 2. Guidelines for using custom protein arrays for protein:protein and protein:DNA interactionstudies are provided in Sections 5 and 6, respectively.

Materials To Be Supplied By the User(Buffer compositions are provided in Section 10.B.)Protein Expression and Verification Reagents• HaloTag® fusion DNA template (see Section 10.A for recommendations)

Note: HaloLink™ Protein Array Systems are recommended for use with HaloTag® Protein encoded by the HaloTag® Flexi® Vectors for optimal results.

• Cell-free expression system (if using the HaloLink™ Array Six Slide System (Cat.# G6190)• HaloTag® TMR Ligand, 5µM (Cat.# G8251, G8252)

Note: The HaloTag® TMR Direct Ligand (Cat.#G2991) is too dilute for this application.)• SDS-PAGE gels and gel-loading buffer (see Section 10.B)

4. Protocol for Expression and Capture of HaloTag® Fusion Proteins to Create a Custom Protein Array(continued)

HaloLink™ Slide Processing Reagents and Equipment

• Bovine serum albumin (Blot-Qualified BSA, Cat.# W3841)• 50ml polypropylene tube and centrifuge for 50ml tubes• 250ml phosphate buffered saline (PBS)• wash bottle• fluorescent slide scanner• dilution buffer (PBSB; See Section 10.B) • wash buffer (PBSI; See Section 10.B; Wash buffer formulations with different salts or detergents can

be used to minimize nonspecific binding and associated fluorescent background.)• fluorescent dye labeled anti-rabbit secondary antibody. Choice of fluorescent dye will depend on

the scanner. Commonly used fluorescent dyes are Cy®3 and Alexa Fluor® 532 for excitation using532nm laser or Alexa Fluor® 633, Alexa Fluor® 647 and Cy®5 for excitation using 635nm red laser.

Figure 2. Overview of experimental workflow for creating a custom protein array.


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HaloTag® Vectors(with appropriate coding region inserted;

provided by the user)

Express HaloTag® fusion proteinsin cell-free expression system

(Section 4.A)

Immobilize HaloTag® fusionproteins on HaloLink™ Slides to

create custom array (Section 4.D)

Attach gasket to HaloLink™ Slideand create humidity chamber

(Section 4.C)

Optional: Confirm expression onSDS-PAGE using HaloTag® TMR labeling (Section 4.B)

Optional: Add replicate sample toconfirm HaloTag® fusion protein

binding using Anti-HaloTag® pAb (Section 4.E)

Use custom array for protein:protein interaction (Section 5) or protein:nucleic

acid interaction (Section 6)

Optional: Add dilution series of HaloTag® standard

protein for normalization(Section 4.F)

Use Anti-HaloTag® pAb to confirmbinding of the HaloTag® fusion

protein (Section 4.E) or fornormalization (Section 4.F)

4.A. Expressing HaloTag® Fusion Proteins

Notes:1. Depending on your HaloTag® fusion protein, you may need to optimize expression.

Solubility, proper folding, and expression levels are some of the factors that may need to beoptimized. See reference 4 for information on factors to consider when expressing proteinssuch as membrane proteins.

2. Five microliters/well of cell-free reaction is added to each well of the HaloLink™ Slide (eachslide contains 50 wells). Scale the cell-free reaction volume to accommodate the number ofassays you wish to perform.

4.A.1. Expression of HaloTag® fusion proteins in the TNT® T7 Quick Coupled Reaction

1. Remove the TNT® T7 Quick Master Mix from storage at –70ºC. Rapidly thaw the Master Mixby hand warming and immediately place it on ice. Other components can be thawed at roomtemperature and stored on ice.

2. Assemble the reaction components in a 0.5ml or 1.5ml microcentrifuge tube as indicated in thetable below.

3. Mix gently by pipetting. If necessary, centrifuge briefly to bring the liquid to the bottom of thetube.

4. Incubate the reaction at 30ºC for 60–90 minutes.

5. Proceed with capture of the protein onto the HaloLink™ Slide (go to Section 4.C). Note: We recommend that you first confirm expression of your HaloTag® fusion protein byanalyzing an aliquot of the reaction by Western blot or fluorescent labeling (see Section 4.B).Store the remainder of the reaction at –70°C while you confirm expression.

6. For long-term storage, we suggest storing expression reactions at –70°C.Note: The stability of expressed proteins in frozen reactions will vary based on the particularprotein involved (5).


Compound VolumeTNT® T7 Quick Master Mix 40µlMethionine 1µlDNA Template (1µg) Xµl

Nuclease-Free Water to a final volume of: 50µl

4.A.2. Expression of HaloTag® fusion Protein in the TNT® SP6 High-Yield Wheat GermReaction

1. Remove TNT® SP6 High-Yield Wheat Germ Master Mix from storage at –70ºC. Rapidly thawthe Master Mix on ice or by hand warming and immediately place on it ice.

2. After the Master Mix has thawed, gently mix several times by pipetting.

3. Assemble the reaction components in a 0.5ml or 1.5ml microcentrifuge tube based on thevolumes provided in the table below.

4. Gently mix after all components are added to the reaction tube.

5. Incubate the translation reaction at 25°C for 2 hours.

6. Proceed with capture of the protein onto the HaloLink™ Slide (go to Section 4.C). Note: We recommend that you first confirm expression of your HaloTag® fusion protein byanalyzing an aliquot of the reaction by Western blot or fluorescent labeling (see Section 4.B).Store the remainder of the reaction at –70°C while you confirm expression.

7. For long-term storage we suggest storing expression reactions at –70°C.

Note: The stability of expressed proteins in frozen reactions will vary based on the particularprotein involved (5).


Compound VolumeTNT® SP6 High-Yield Wheat Germ Master Mix 30µlDNA Template (8µg) Xµl

Nuclease-Free Water to a final volume of: 50µl


4.B. Confirming Expression of HaloTag® Fusion Proteins with an SDS-Gel (optional)

Before creating custom arrays, we recommend that you confirm expression of your HaloTag® fusionproteins by labeling with fluorescent HaloTag® TMR ligand (not supplied; Cat.# G8251, G8252) followedby SDS-PAGE analysis. Western blotting using the supplied Anti-HaloTag® pAb and an appropriatelylabeled anti-rabbit secondary antibody can also be used to confirm expression. A dilution series of theHaloTag® Standard Protein can be run in parallel to estimate the expression level of your HaloTag®

fusion protein. Typical dilution range of HaloTag® Standard Protein for a TNT® T7 Quick Reaction is1.0–10µg/ml (0.016–0.16nmol/ml) for a TNT® SP6 High-Yield Wheat Germ Reaction it is 10–100µg/ml(0.16–1.6nmol/ml).

1. Prepare a 500-fold dilution of HaloTag® TMR Ligand stock solution (5mM) in PBS for a finalconcentration of 10µM. Dilute enough ligand to accommodate the number of samples youwish to analyze.

2. Mix 2.0µl of cell-free reaction containing the HaloTag® fusion protein with 1.0µl of HaloTag®

TMR Ligand (10µM).

3. Add 7.0µl of PBS to a final volume of 10.0µl. Incubate at room temperature, protected fromlight, for 30 minutes.

4. Remove 5.0µl of the reaction, add 5.0µl of 2X SDS gel loading buffer and heat to 70°C for 2 minutes. Separate on an SDS-polyacrylamide gel. Analyze on a fluorescent scanner.

6. Scan the gel using a fluorescent scanner (e.g., Typhoon®, Amersham Biosciences, Ltd.,555nmEx; 585nmEm). The HaloTag® fusion protein will appear as a fluorescent band on the gel(Figure 3). If a dilution series of the HaloTag® Standard Protein was used, HaloTag® fusionprotein expression levels can be estimated by comparing the band intensity with those of thestandard samples.

Figure 3. HaloTag® fusions were expressed in a TNT® T7 Quick Reaction, labeled with HaloTag®-TMR Ligandand analyzed on a SDS gel. Lane M: molecular weight markers, lane 1: HaloTag®-cJun, lane 2: HaloTag®-cFos, lane3: HaloTag®-PKA, lane 4: HaloTag®-R1�, lane 5: HaloTag®-p53, lane 6: HaloTag®-p65, lane 7: HaloTag®-�Gal, lane 8:HaloTag® Protein alone, no fusion (specificity control).

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M 1 2 3 4 5 6 7 8

155

98

63

40

32

21

kDa

4.C. Attaching the HaloLink™ Array Gasket to the HaloLink ™ Slide

Important! Read all the instructions before attempting to attach the slides to the gaskets.

Precautions for Handling Slides:

• Work in a dust-free environment and always wear powder-free gloves. Dust and particlesfrom powdered gloves can interfere with scanning.

• Only one side of the slide is coated. The coated side is the one from which you can read thebar code number correctly.

• Do not touch the coated slide surface, and hold slides only at the edges.

• Equilibrate the slide box to ambient temperature before taking a slide out. Any remainingslides can be returned to –20°C for up to one month.

• Only one side of the HaloLink™ Array Gasket is sticky. The gasket is sandwiched between aclear and an opaque plastic sheet. The sticky side is the side covered with the clear plasticsheet.

• Do not remove an attached gasket and then reattach it to the slide. If the gasket misaligns, useanother gasket and slide. Do not reuse a slide on which a gasket has been misaligned.

• All experiments on HaloLink™ Slides must be performed in a humidity chamber. A methodfor making a humidity chamber is provided in the protocol below.

Protocol


1. Humidity chambers are needed for HaloLink™ Slide experiments.

a. Cut or fold 4 or 5 paper towels into approximately 5 × 5 inch squares andsoak them in water. Place them on the lab bench.

b. Cover the towels with a plastic lid to form a humidity chamber. (A lidfrom a 1.0ml pipette tip box works well.) Humidity inside the box will beapproximately 90%.

!


2. Remove the gasket from the bag, and place it on the lab benchwith the clear plastic sheet facing up.

3. Remove the top clear plastic sheet by holding down the gasket atone corner and gently peeling off the clear plastic sheet with theother hand.

4. Remove the HaloLink™ Slide from the box, being sure to holdthe slide at the edges. Face the coated side toward the bluegasket. The coated side is the one from which you can read thebar code number correctly.

5. Hold the slide at an approximately 45° angle with the long edgeof the slide aligned with the edge of the gasket. Make sure thatthe gasket is in the center of the slide.

6. Lower the other edge of the slide carefully over the gasket. Thelong edge of the gasket should not extend beyond the glass slide.


7. Press the back of the slide and along the edges of the slide tomake a water-tight seal between the gasket and the glass.

8. Pick up the glass slide with the gasket attached.

9. Look at the slide from the back and make sure no air bubbles aretrapped. Air bubbles will cause leaks. Trapped air bubbles can beremoved by placing the slide gasket-side-down on the opaqueplastic sheet and gently pressing down on the back of the glassslide.

10. Place the opaque plastic sheet on the wet paper towel in thehumidified chamber.

11. Place the HaloLink™ Slide on the plastic sheet, gasket-side-up.

12. Cover with a plastic lid.

4.D. Immobilization of HaloTag® Fusion Proteins on HaloLink™ Slides

A custom protein array is made by adding the cell-free reaction containing the expressed HaloTag®

fusion protein into individual wells on the slide. The HaloTag® fusion protein binds to the HaloLink™Slide without any need for prior purification.

Controls

1. Capture Controls should be performed to confirm capture of HaloTag® fusion proteins on theslide. Dedicate one replicate well as a control for each fusion protein being analyzed. Captureof HaloTag® fusion proteins can be confirmed by probing with the supplied Anti-HaloTag®

Antibody, followed by detection with a labeled anti-rabbit secondary antibody (see Section4.E for details).

2. Normalization Control: The HaloTag® Standard Protein (provided with Cat.# G6140 andG6180) can be used to normalize for variations in expression level and capture efficiencybetween different HaloTag® fusion proteins. A concentration series of the HaloTag® StandardProtein can be used to estimate the amount of HaloTag® fusion protein expression andcapture for normalization purposes (see Section 4.F for details).

3. Specificity Control: HaloTag® Protein expressed without an attached fusion can serve as acontrol to evaluate specificity of interactions in downstream applications. A protocol forcreating a construct that expresses only HaloTag® Protein can be obtained by calling technicalservices.

4. Additional controls may be needed for downstream applications (e.g., protein:protein orprotein:DNA interaction studies). We suggest including an Extract Control (extract that doesnot contain expressed protein) to evaluate nonspecific binding of proteins in the translationlysate. We also suggest including a PBSB-only control to evaluate background, plusappropriate known binding partners as positive controls.

Warning

• Maintaining the conformational integrity of captured proteins is essential for successfuldownstream applications using custom arrays. The characteristics of each HaloTag® fusionprotein will depend on the individual protein that is fused to the HaloTag® protein. You willneed to optimize the conditions for your individual custom array. For example, proteinssubject to denaturation during slide washing and drying may be stabilized by adding sucrose,trehalose or glycerol to the wash buffer (5).

• We recommend making the custom array and performing the downstream application on thesame day. Long-term stability of custom arrays depends upon the characteristics of the boundprotein fusion and will need to be determined for each custom array (5).

• The HaloLink™ Slide is coated with a unique hydrogel that can be damaged if it is scratchedby pipette tips. Use extreme care when pipetting samples into the wells. During pipetting,keep the pipette tip just above the slide surface. Support the pipette tip on the gasket edge,and keep it above the slide surface.


!


Protocol

1. Prepare samples for HaloLink™ Slide experiment and apply to wells.

a. If you have frozen your cell-free expression reactions, bring them to room temperaturebefore beginning this procedure. Pipet 5.0µl of cell-free reaction (from Section 4.A.1 or4.A.2) containing the HaloTag® fusion proteins into the wells. We recommend preparingreplicate wells for each sample. One replicate well may be used to confirm HaloTag®

immobilization (Section 4.E). Additional replicate wells may be necessary for downstreamapplications.

b. If you are planning to normalize the data, prepare a concentration series of HaloTag®

Standard Protein (see Section 4.F) in parallel.

c. Include appropriate controls (e.g., extract control, PBSB-only control). You may also needto design additional controls specific for your experiment.

2. Incubate the slide in the humidity chamber for 1 hour at room temperature.

3. Following incubation, hold the slide at a 45º angle, and wash the wells with a gentle stream ofwash buffer (PBSI) using a wash bottle.

Note: Do not forcefully squirt the wash buffer directly onto the glass surface because youmay damage the coating.

4. Fill a 50ml polypropylene tube with 40ml of PBSI. Place the slide into the 50ml tube, cap thetube, and wash the slide by inverting the tube 10 times.

5. Drain the wash buffer and dry the slide by centrifuging the tube for 3 minutes at 350 × g atroom temperature.

6. Take the slide out of the tube using plastic tweezers (do not scratch the surface), andimmediately place it in the humidity chamber. Overdrying the slide may lead to proteindenaturation.

7. The slide is now ready for use in your downstream application (Sections 5 and 6 provideguidelines for protein:protein and protein:DNA interaction applications, respectively).

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4.E. Confirming Capture of HaloTag® Fusion Proteins on HaloLink™ Slides (optional)

We recommend that you confirm binding of your HaloTag® fusion proteins to the HaloLink™ slide. Useone replicate well from Section 4.D for this step. This step should be performed at the same time asyour chosen downstream application.

1. Dilute the Anti-HaloTag® pAb 1:200 in PBSB to give a final concentration of 5.0µg/ml. Youwill use 8µl of this solution per well. Prepare sufficient volume for the number of wells used.

2. Add 8.0µl/well of Anti-HaloTag® pAb to wells containing HaloTag® fusion proteins. Performno-protein control by adding 8.0µl of the diluted antibody solution to a well containing noHaloTag® protein. Perform an extract control by adding diluted antibody solution to wellscontaining extract that does not contain expressed protein.

3. Incubate for 1 hour at room temperature in the humidified chamber.

4. Following incubation, hold the slide at a 45º angle and, using a wash bottle, wash the wellswith a gentle stream of wash buffer (PBSI). Note: Do not forcefully squirt wash buffer directly onto the glass surface because this maydamage the coating.

5. Fill a 50ml polypropylene tube with 40ml of PBSI. Place the slide into the 50ml tube, cap thetube and wash the slide by inverting the tube 10 times.

6. Drain the wash buffer, and dry the slide by centrifuging the tube for 3 minutes at 350 × g atroom temperature.

7. Take the slide out of the tube using plastic tweezers, and immediately place it in the humiditychamber.

8. Dilute fluorescent-dye-labeled, anti-rabbit secondary antibody in PBSB. 10µg/ml is a goodstarting concentration; however, the concentration may have to be optimized for individualsecondary antibodies.

9. Add 8.0µl of the diluted anti-rabbit secondary antibody to wells incubated withAnti-HaloTag® pAb.Note: Concentration and volume of secondary antibody will vary. A good starting point is8.0µl of a 10µg/ml secondary antibody.

10. Incubate for 1 hour in the humidity chamber, protected from light (cover the chamber in foil).

11. Wash the slides as described in Steps 4.

12. Remove the gasket using tweezers. Once removed, the gasket can not be reattached.

13. Wash and spin dry the slides as descried in Steps 5 and 6.

14. Scan the slides. See Section 7 for details. The stability of the slides will depend on the dyeused with the secondary antibody. Follow the recommendations of the dye manufacturer. Inour experience, slides that are protected from light may be stored overnight before scanning.


!

4.F. Using HaloTag® Standard Protein for Normalization (optional)

We recommend that you normalize your experiment to account for differences in expression and capture ofvarious HaloTag® fusion proteins. This experiment can be done on the same slide used for the capture ofHaloTag® fusion proteins and downstream application or on a separate slide depending on the number ofsamples being processed.

1. Prepare a dilution series of HaloTag® Standard Protein in PBSB. A range of 0.1–10µg/ml is a goodstarting point. Use PBSB alone containing no HaloTag® Standard Protein as a blank.

2. Add 5.0µl of each concentration of HaloTag® Standard Protein into individual wells on theHaloLink™ Slide. For the blank, use 5.0µl of PBSB.

3. Dilute the cell-free expression reaction in PBSB to bring the concentration of the HaloTag® fusionproteins within the concentration range of the HaloTag® Standard Protein (0.1–10.0µg/ml). Typicalexpression level of HaloTag® fusion protein is 1.0–10.0µg/ml in TNT® T7 Quick Reaction and10–100µg/ml in TNT® SP6 High-Yield Wheat Germ Reaction. Note: Run on an SDS gel with the TMR Ligand for detection or make 2 to 3 dilutions to be sure thatat least one falls within the calibration range,

4. Add 5.0µl of each diluted cell-free expression reaction sample into a separate well on the HaloLink™Slide.


6. Following incubation, hold the slide at a 45º angle and, using a wash bottle, wash the wells with agentle stream of wash buffer (PBSI). Note: Do not forcefully squirt wash buffer directly on the glass surface because this may damage thecoating.

7. Fill a 50ml polypropylene tube with 40ml of PBSI. Place the slide into the 50ml polypropylene tube,cap the tube and wash the slide by inverting the tube 10 times.

8. Drain the wash buffer, and dry the slide by centrifuging the tube for 3 minutes at 350 × g at roomtemperature.


10. Dilute the Anti-HaloTag® pAb 1:200 in PBSB to give a final concentration of 5.0µg/ml. You will use8µl of this solution per well. Prepare sufficient volume for the number of wells used.

11. Add 8.0µl/well of Anti-HaloTag® pAb to wells containing HaloTag® fusion proteins. As a no-proteincontrol, add 8.0µl of the diluted antibody solution to wells containing no HaloTag® protein. Performextract control by adding diluted antibody to wells containing extract that does not containexpressed protein.

12. Incubate for 1 hour at room temperature in the humidity chamber.

13. Wash and spin-dry the slides as described in Steps 6–9.


!


14. Dilute fluorescent-dye-labeled, anti-rabbit secondary antibody in PBSB. 10.0µg/ml is a goodstarting concentration; however, the concentration may have to be optimized for individualsecondary antibodies. You will use 8µl of this solution per well. Prepare sufficient volume forthe number of wells used.

15. Add 8.0µl of the diluted anti-rabbit secondary antibody to the wells incubated withAnti-HaloTag® antibody.

16. Incubate for 1 hour protected from light at room temperature in the humidity chamber (e.g,cover the chamber with foil).

17. Wash the slides as described in Step 6.

18. Carefully remove the gasket using tweezers. Once removed, the gasket cannot be reattached.

19. Wash and spin dry the slides as described in Steps 7 and 8.

19. Scan the slides (see Section 7 for details). The stability of the slides will depend on the dyeused with the secondary antibody. Follow the recommendations of the dye manufacturer. Inour experience, slides protected from light may be stored overnight before scanning.

5. Guidelines for Protein:Protein Interaction Analysis

Custom arrays containing a panel of HaloTag® fusion proteins can be interrogated with a probe proteinto identify key protein:protein interactions. Probe proteins can be obtained from several sourcesincluding cell-free systems, purified proteins and cell lysates. This manual contains guidelines forperforming protein:protein interaction studies using a probe protein expressed in a cell-free system(Figure 4). The example protocol provided is divided into two phases: 1) the probe protein is expressed ina cell-free expression system and labeled in vitro with biotin during expression; and 2) the biotinylatedprobe protein is added to the custom array on the HaloLink™ Slide, and protein:protein interactions aredetected by adding fluorescently labeled streptavidin. Optimization of protein:protein interaction andwashing conditions may be required for your specific HaloTag® fusion and probe protein pair (5).

Phase 1: Expression of Probe Protein. The probe is the protein used to interrogate the array slidecontaining the expressed HaloTag® fusion protein. Probe protein is expressed in a cell-free system andlabeled by incorporating biotinylated lysine residues into the nascent protein during in vitro translationusing Transcend™ Non-Radioactive Translation Detection System (Cat.# L5080, L5070). See TechnicalBulletin #TB182 for instructions for using this system (available online at: www.promega.com/tbs/)

Figure 4. Schematic of HaloTag® fusion and probe protein interaction analysis on HaloLink™ Slides. There areseveral methods for expressing, binding and detecting probe protein interactions. This schematic uses expressionand labeling of probe protein in a cell-free system for illustration purposes.


7890

MA


HaloTag® fusionproteins

Specificitycontrol

(if required)

B

BB

B

Biotinylated probe

Phase 1Cell-free expression of

biotinylated probe

Phase 2Probe capture

Incubate custom arraywith biotinylated prey

Fluorescent signal No signal

Wash and spin dry

Detect positiveinteraction with

Streptavidin-Fluor

HaloTag® protein

Fused protein

HaloLink™ Slide well coated with HaloTag® ligand

Streptavidin-Fluor

5. Guidelines for Protein:Protein Interaction Analysis (continued)

Phase 2: Capture of Probe Protein

Biotinylated probe protein is added to the custom protein array created on HaloLink™ Slides. PositiveHaloTag® interaction is detected using fluorescently labeled streptavidin. Depending on the protein pairinvolved, different degrees of optimization of binding conditions may be required. The addition ofcofactors, salts or detergents may be required for certain protein interactions. It is important to considerthe following points before designing any protein:protein interaction experiment:a. Affinity of interactionb. Concentration of probe proteinc. Functional conformation of HaloTag® fusion protein and probe proteind. Detection method used for interactionsAppropriate controls should be included in the experiment, including Extract Controls, SpecificityControls, and Normalization Controls (see Section 4.D).

5.A. Expression and Cotranslational Labeling of Probe Protein in a Cell-Free System

Materials to be Supplied by the User • custom protein array on HaloLink™ Slide (from Section 4.D)• DNA template for expressing probe protein• Transcend™ Non-Radioactive Translation Detection System (Cat.# L5080, L5070)• cell-free expression system: TNT® T7 Quick or TNT® SP6 High-Yield Wheat Germ System• fluorescent dye-labeled streptavidin. (Choice of fluorescent dye will depend on the scanner.)Commonly used fluorescent dyes are Cy®3 and Alexa Fluor® 532 for excitation using a 532nm laser orAlexa Fluor® 633, Alexa Fluor® 647 and Cy®5 for excitation using a 635nm red laser.Note: 5µl/well of a cell-free reaction containing probe protein is added to each well of the customHaloLink™ Slide. Scale up the reaction volume to accommodate the number of replicates and assaysyou wish to perform.

5.A.1. Expression of Probe Protein in the TNT® T7 Quick Reaction

1. Remove the TNT® T7 Quick Master Mix from storage at –70ºC. Rapidly thaw the Master Mixby hand-warming and immediately place on ice. Other components can be thawed at roomtemperature and stored on ice.

2. Assemble the reaction components in a 0.5ml or 1.5ml microcentrifuge tube based on thevolumes provided below.


Compound Volume

TNT® T7 Quick Master Mix 40µlMethionine 1µl

DNA Template (0.2–2µg) Xµl

Transcend™ tRNA 1–2µlNuclease-Free Water to a final volume of: 50µl

5.A. Expression and Cotranslational Labeling of Probe Protein in a Cell-Free System (continued)

5.A.1. Expression of Probe Protein in the TNT® T7 Quick Reaction (continued)

Note: 5µl/well of cell-free reaction containing probe protein is added to each well of thecustom HaloLink™ Array Slide. Scale up the reaction volume to accommodate the number ofreplicates and assays you wish to perform.

3. After adding of all the components, gently mix by pipetting. If necessary, centrifuge briefly toreturn the reaction to the bottom of the tube.

4. Incubate the reaction at 30ºC for 60–90 minutes.

5. Optional: Confirm the expression for biotin labeled probe protein using Western blot (seeTranscend™ Non-Radioactive Translation Detection Systems Technical Bulletin #TB182).

5.A.2. Expression of Probe Protein in the TNT® SP6 High-Yield Wheat Germ Reaction (Phase I)

1. Remove the TNT® SP6 High-Yield Wheat Germ Master Mix from storage at –70ºC. Rapidlythaw the Master Mix on ice or by hand-warming and immediately place on ice.

2. After the Master Mix has thawed, gently mix several times with a pipette tip or by pipetting.

3. Assemble the reaction components in a 0.5ml or 1.5ml microcentrifuge tube based on thevolumes provided below.

Note: 5µl/well of a cell-free reaction containing probe protein is added to each well of theHaloLink™ Slide. Scale the reaction volume to accommodate the number of replicates andassays you wish to perform.

4. Mix gently after all components are added to the reaction tube.

5. Incubate the translation reaction at 25°C for 2 hours.

6. Optional: Confirm the expression for biotin-labeled prey protein using Western blot (seeTranscend™ Non-Radioactive Translation Detection Systems Technical Bulletin #TB182).


Compound Volume

TNT® SP6 High-Yield Wheat Germ Master Mix 30µl

DNA Template (2–12µg) Xµl

Transcend™ tRNA 1–2µlNuclease-Free Water to a final volume of: 50µl


5.B. Protein:Protein Interaction Analysis

In phase 2, the biotinylated probe protein is incubated with the custom protein array created asdescribed in Section 4.D.

1. Add 5.0µl of cell-free expression reaction containing probe protein to the wells containingimmobilized HaloTag® fusion protein. Add probe to appropriate control wells (Section 4.D).


3. Following incubation, hold the slide at a 45º angle, and wash the wells with a gentle stream ofwash buffer (PBSI) using a wash bottle. Note: Do not forcefully squirt wash buffer directlyonto the glass surface because this may damage the coating.

4. Fill a 50ml polypropylene tube with 40ml of wash buffer. Place the slide into the tube, cap it,and wash the slide by inverting the tube 10 times.

5. Drain the wash buffer, and dry the slide by centrifuging the tube for 3 minutes at 350 × g atroom temperature.


7. Detection Reagent: Fluorescent-dye-labeled streptavidin is used for detecting protein:proteininteractions. The optimal concentration will have to be determined by the user; however, aconcentration of 10.0 µg/ml labeled streptavidin in PBSB is a good starting point.

8. Add 8.0µl of dye-labeled streptavidin to each well containing immobilized HaloTag® bait andnegative controls.

9. Incubate for 1 hour protected from light at room temperature in the humidity chamber (coverthe chamber with foil).

10. Wash the slides as described in Step 3, and remove the gasket using tweezers.

11. Place the slide into a 50ml tube, and fill it with 40ml of wash buffer. Cap the tube and washthe slide by inverting the tube 10 times.

12. Drain the wash buffer, and dry the slide by centrifuging for 3 minutes at 350 × g at roomtemperature.

13. Scan the slide (see Section 7 for details).

6. Guidelines for Protein:DNA Interaction Analysis

Custom arrays containing panels of HaloTag® fusion proteins can be queried with DNA probes toidentify key protein:DNA interactions. Key points to consider before performing protein:DNA interaction analysis are: a. Affinity of interactionb. Concentration of probe DNAc. Functional conformation of HaloTag® fusion proteins and DNA probed. Method used to detect positive interactions

6. Guidelines for Protein:DNA Interaction Analysis (continued)

A schematic for performing protein:DNA interaction analysis on HaloLink™ Slides is shown in Figure 5. The protocol is divided into two phases:Phase 1: 5´ Labeling of the DNA probe with fluorescent dye. The 5´ end-labeled DNA used for the probeshould be of the highest purity. 5´ end-labeled nonspecific DNA can be prepared as a specificity controlfor HaloTag® fusion protein:probe interaction. Starting concentration of DNA will depend on specificprotein:DNA interaction. For an initial experiment, we suggest running a titration series to optimize theDNA concentration. Add 5-10µl/well of labeled DNA probe to each well of the HaloLink™ Slide. Scalethe reaction volume to accommodate the number of assays you wish to perform.

Phase 2: DNA Capture. The DNA probe is added to custom arrays on the HaloLink™ Slide.Appropriate controls should be included in the experiment (e.g., extract control, specificity control andnormalization controls). See Section 4.D for more information.

Figure 5. Schematic of DNA:protein interaction analysis on HaloLink™ Slides.Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TM310 Printed in USA.Page 24 Revised 12/15


HaloTag® fusionproteins

Specificitycontrol

(if required)

Fluorescent dye-labeledDNA

Phase 1Synthesis of DNA probe

Phase 2Probe capture

Incubate custom arraywith DNA

Fluorescent signal No signal

Wash and spin dry

HaloTag® protein

Fused protein

HaloLink™ Slide well coated with HaloTag® ligand


DNA Capture on the HaloLink™ Slide

This protocol uses DNA labeled with fluorescent dye to probe proteins immobilized on HaloLink™ slides. Theprotocol provides general guidelines for protein:DNA interaction assays. Optimization of conditions for yourspecific protein:DNA interaction may be required.

Materials to be Supplied by the User• DNA labeled with fluorescent dye at the 5´ end. Labeled DNA should be of highest purity.• custom protein array on a HaloLink™ Slide (Section 4.D)

1. Dilute the DNA in PBS. The final DNA concentration used will depend on the individual protein-DNA interaction and will have to be optimized. Note: As an example, in an experiment with a HaloTag®-p65 fusion, 100fmol/µl of labeled DNAproved to be optimal. For the competition experiments for p65 binding, cold DNA was titrated into100fmol/µl of labeled DNA at the following ratios (labeled to unlabeled) 1:0, 1:0.1, 1:0.5, 1:1, 1:3, 1:6,1:20, 1:40, 1:80.

2. Add 5.0µl of labeled DNA to wells containing immobilized HaloTag® fusion protein.

3. Incubate the slide at room temperature for 1 hour protected from light in the humidity chamber(cover the chamber with foil).

4. Following incubation, hold the slide at a 45º angle and, using a wash bottle, wash the wells with agentle stream of wash buffer (PBSI). Do not forcefully squirt wash buffer directly on the glass surfaceas it may damage the coating.

5. Remove the gasket using tweezers.

6. Fill a 50ml polypropylene tube with 40ml of PBSI. Place slide into the tube, cap it, and wash the slideby inverting the tube 10 times.

7. Drain the wash buffer, and dry the slide by centrifuging the tube for 3 minutes at 350 × g at roomtemperature.

8. Scan the slide (see Section 7 for details).

7. Scanning and Data Analysis of HaloLink™ Slides

7.A. Scanning

Suggestions below for scanning and data analysis of HaloLink™ Slides are made based on our use of aGenepix® 4000B scanner. However, any other conventional microarray scanner can be used, provided that it canaccept 1 x 3-inch slides and is compatible with the fluorescent dye used on the slide.

1. Insert the slides face-down in the scanner.

2. Adjust the laser and photomultiplier tube (PMT) setting based on the fluorescent dye used in theexperiment.

3. If the dye is photosensitive, use a lower laser power to avoid photobleaching.

4. We recommend doing a preview scan to identify whether the detector and laser settings are set tomaximize dynamic range.

5. Scan the slide at 10µm resolution.

6. For data analysis, use the scanner software to calculate the median fluorescence intensity from a2.0mm diameter area in the center of each well.

7.B. Data Analysis

Data Normalization

To obtain reproducible results from protein array experiments, you need to control for experimental andrandom variations. Experimental factors that will introduce variability in HaloLink™ Protein Array include:

1. Variations from expression and capture of HaloTag® fusion proteins:a. Differences in expression levels of various HaloTag® fusion proteinsb. Day-to-day and batch-to-batch variation in expression level of a single HaloTag® fusion proteinc. Differences in protein expression based on the choice of protein expression systems d. Variation in capture efficiency of different HaloTag® fusion proteins

2. Variations coming from specific probe binding to HaloLink™ Protein Arraysa. Source of probe generation; for example, protein probes can be expressed in cell-free systems

(Section 5) or in cells or purified protein. b. Detection methodc. Binding affinity

Random variations may be introduced by the operator and may depend on reagents (wash buffer, slideprocessing steps) as well as time and place of experiments.

HaloTag® Standard Protein can be used to normalize for the difference in expression and capture levels ofvarious HaloTag® fusion proteins (Sections 4.B. and 4.F). For normalization as described in Section 4.F., preparea standard curve by plotting the blank corrected median RFU values against concentration in µg/ml for theHaloTag® Standard Protein. Use the standard curve to calculate the concentration of the diluted HaloTag®

Fusion proteins. Multiply the concentration determined from the standard curve by dilution factor to get theexpression level in the cell-free expression system. To normalize the results from downstream application forvariations in expression level and capture, fluorescent signal from protein-interaction studies can be divided byamount of protein calculated from standard curve (6).

There are several excellent articles on normalization of microarray data that can be adapted for HaloLink™Protein Array Systems (7–9).



8. Troubleshooting

For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information available at:www.promega.com. E-mail: [email protected]

Symptoms Causes and Comments

HaloTag® fusion protein not detected DNA quality was poor, or wrong DNA quantity was used. Use only highly purified plasmid DNA. with SDS gel using HaloTag® TMR We strongly recommend that you refer to the TNT® T7 Quick Coupled Transcription/Ligand Translation Systems Technical Manual #TM045 or the TNT® SP6 High-Yield Wheat Germ Protein

Expression System Technical Manual # TM282 for specific details on use of these expressionsystems.

Fluorescent scanner does not have appropriate filters and/or sensitivity. Use a fluorescentscanner with appropriate sensitivity (e.g., Typhoon®, GE Healthcare). For scanners with lowersensitivity, sample volume may need to be increased.

Samples may have been overheated. Reduce the denaturation temperature to 60°C for5–10 minutes.

Gasket not sticking to the glass Only one side of the gasket is sticky. Make sure the sticky side is toward the glass slide.

Gasket came off of the glass during Make sure the gasket does not extend beyond the edges of the slide. centrifuging in 50ml tube

No signal or weak signal with Wrong secondary antibody used. Use an anti-rabbit secondary antibody labeled with fluorescent HaloTag® Standard Protein dye.

Incorrect scanner setting. Make sure the laser and photomultiplier tube (PMT) setting matches the fluorescent dye used for detection.

Photobleaching. Avoid prolonged exposure of the fluorescent-dye-labeled secondary antibody tolight. Avoid multiple scans of the slide.

No signal or weak signal when HaloTag®-fusion protein not expressed or poorly expressed. Confirm HaloTag®-fusion protein HaloTag®-fusion protein is probed expression by HaloTag®-TMR labeling or Western blot. using Anti-HaloTag® pAb

Assay not performed correctly. Confirm that HaloTag® Standard protein gives a positive signal.

Incorrect scanner setting. Make sure the laser and photomultiplier (PMT) setting matches thefluorescent dye used for detection.

Scan shows dark area or areas Scratches on slide surface. The slide is coated with a thin hydrogel layer that can be scratchedwith very bright spots within during processing (pipetting, washing, centrifuging). Be careful not to touch the slide surfacethe wells during the assay and downstream processing steps.

No signal after protein:protein No or low expression of probe protein. Confirm expression of the probe protein by Western blot.interaction experiment Optimize conditions to maximize expression of probe protein.

No incorporation of biotin during prey protein expression. Biotin incorporation is proteindependent. Confirm biotin incorporation by Western blot.

Weakly interacting HaloTag® fusion and probe pairs may dissociate during a multistep assay.Optimize washing and incubation steps to minimize dissociation.

8. Troubleshooting (continued)

Symptoms Causes and Comments

No signal after protein:DNA Low incorporation of fluorescent dye in DNA. Check incorporation of the fluorescent dye.interaction analysis

Increase the concentration of the starting DNA solution.

Binding and wash buffers may need to be optimized for each protein:DNA pair.

Weakly interacting protein:DNA pairs may dissociate during a multistep assay. Optimizewashing and incubation steps to minimize dissociation.

High background; Insufficient washing. Follow the proper washing procedure to minimize background.

high background fluorescence Nonspecific binding. HaloTag® protein binding to the HaloLink™ Slide is covalent. Hence non-specific binding during capture of HaloTag® fusion protein from a cell-free system can bereduced by increasing the stringency of washing. Add detergent and/or higher concentration of salt to the wash buffer. Increase the number or duration of washes.

9. References

1. Los, G. et al. (2005) HaloTag® interchangeable labeling technology for cell imaging and protein capture. Cell Notes 11, 2–6.

2. Los, G. and Wood, K. (2007) The HaloTag: A novel technology for cell imaging and protein analysis. Methods. Mol. Biol. 356, 195–208.

3. Los, G. et al. (2008) HaloTag: A novel labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 6, 373–82.

4. Arduengo, M., Schenborn, E. and Hurst. R. (2007) The role of cell-free rabbit reticulocyte expression systems in functional proteomics.In: Cell-Free Expression. Kudlicki, W., Katzen, F. and Bennett, R.P., eds. Landes Bioscience.

5. Nath, N. (2008) Improving Protein Array Performance: Focus on Washing and Storage Conditions. J. Proteome Res. 7, 4475–82.

6. Olle, E.W. et al. (2005) Development of an internally controlled antibody microarray. Mol. Cell. Proteomics 4, 1664–72.

7. Shi, L. et al. (2008) Reproducible and reliable microarray results through quality control: good laboratory proficiency and appropriatedata analysis practices are essential. Curr. Opin. Biotechnol. 19, 10–8.

8. Kreil, D.P. (2005) There is no silver bullet—A guide to low-level data transforms and normalization methods for microarray data. BriefBioinform. 6, 86–97.

9. Ingvarsson, J. (2007) Design of recombinant antibody microarrays for serum protein profiling: targeting of complement protiens. J.Proteome Res. 6, 3527–36.



10. Appendix

10.A. HaloTag® Vectors

The desired protein coding sequence must be cloned into a HaloTag® vector according to the cloningrecommendations supplied with that vector. We recommend using the HaloTag® Flexi® Vectors. The pFN19A orpFN19K can be used to prepare a construct that expresses HaloTag® Protein alone. Contact technical services formore information. Vectors suitable for use with this system include the following:

Product Cat.#pFN19A HaloTag® T7 SP6 Flexi® Vector G1891pFN19K HaloTag® T7 SP6 Flexi® Vector G1841pFC20A HaloTag® T7 SP6 Flexi® Vector G1681pFC20K HaloTag® T7 SP6 Flexi® Vector G1691

Compatibility with Protein Expression Systems

All of the above vectors include both T7 and SP6 promoters and so can be used to express proteins in either theTNT® T7 Quick Coupled Transcription Translation System or the TNT® SP6 High-Yield Wheat Germ ProteinExpression System.

Vector Nomenclature

The designation “pFN“ indicates an N-terminal fusion vector, and“pFC“ indicates a C-terminal fusion. Thedesignation“A“ or “K“ in a vector name indicates that the vector carries ampicillin or kanamycin resistance.

Cloning Strategies

Fusing HaloTag® protein at the C- or N- terminal to the protein of interest may impact the expression ofHaloTag® fusion proteins, as well as the downstream application of custom arrays created using these fusions.Please refer to the Flexi® Vector Systems Technical Manual # TM254 (www.promega.com/tbs/tm254/tm254.html)for detailed instructions on cloning into the HaloTag® Flexi Vectors.

10.B.Composition of Buffers and Solutions

1X PBS 1.9mM sodium phosphate monobasic8.1mM sodium phosphate dibasic150mM sodium chloride

Adjust pH to 7.4

4X SDS Gel Loading Buffer0.24mM Tris-HCl (pH 6.8)

3mM bromophenol blue50.4% glycerol0.4M dithiothreitol

2% SDS

PBSB1X PBS

10mg/ml BSA

PBSI1X PBS

0.05% IGEPAL® CA-630 (Sigma Cat# I 3021 or I 8896)

Due to the solubility properties of IGEPAL® CA-630,we recommend that you first prepare a 10% stocksolution in water and use this stock solution toprepare the PBSI wash buffer.

10.C. Related Products

Product Cat.#Anti-HaloTag® pAb G9281HaloTag® Standard Protein G4491HaloTag® TMR Ligand G8252TNT® T7 Quick Coupled Transcription/Translation System L1170 TNT® T7 Quick Coupled Transcription/Translation System, Trial Size L1171 TNT® SP6 High-Yield Wheat Germ Protein Expression System (4 × 300µl) L3260TNT® SP6 High-Yield Wheat Germ Protein Expression System (1 × 300µl) L3261Flexi® Enzyme Blend (Sgf1 and PmeI) R1851

10.D. Summary of Change

The following change was made to the 12/15 revision of this document:Updated licensing and disclaimer statements.



(a)BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the termsof this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.Researcher may use this product for research use only; no transfer or commercial use of this product is allowed. Commercial use means any and all uses of this product by a partyin exchange for consideration, including, but not limited to (1) use in further product manufacture; (2) use in provision of services, information or data; and (3) resale of the productor its derivatives, whether or not such product or derivatives are resold for use in research. With respect to any uses outside this label license, including any commercial,diagnostic, therapeutic or prophylactic uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OFANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THE PRODUCT.The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.(b)Licensed exclusively under U.S. Pat. No. 7,172,905.© 2009, 2015 Promega Corporation. All Rights Reserved.Flexi, HaloTag and TNT are registered trademarks of Promega Corporation. HaloLink and Transcend are trademarks of Promega Corporation.Alexa Fluor is a registered trademark of Molecular Probes, Inc. Cy is a registered trademark of GE Healthcare Bio-sciences. Genepix is a registered trademark of Axon Instruments,Inc. IGEPAL is a registered trademark of Rhone-Poulenc AG, Co. Typhoon is a registered trademark of GE Healthcare Biosciences.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.All prices and specifications are subject to change without prior notice.Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.

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